Detection of actin-binding proteins in human platelets by 125I-actin overlay of polyacrylamide gels.
Actin-binding proteins have been identified in human platelets with a gel-overlay technique that uses 125I-G-actin. Platelet proteins were separated on SDS polyacrylamide gels using the buffer system of Laemmli (1970, Nature [Lond.] 227:680-685). The proteins were fixed in the gels with methanol-acetic acid, the SDS was washed out, and the proteins were renatured. The gels were incubated with 125I-G-actin from rabbit skeletal muscle that was radiolabeled with 125I according to the method of Bolton and Hunter (1973, Biochem. J. 133:529-538) and has been shown to retain biological activity. After nonspecifically bound radioactivity was washed out, gels were dried and processed for autoradiography. The 125I-G-actin binds to several proteins in human platelets, platelet extracts, and the particulate fraction. Control experiments demonstrate that the 125I-G-actin can be displaced by use of increasing amounts of unlabeled actin, that the binding is stable to 0.6 M NaCl, and that preheating the 125I-G-actin to 90 degrees C for 3 min eliminates all binding. Prominent 125I-G-actin-binding activities were present at Mr 90,000 and 40,000. The binding to the 90,000 Mr protein appears to be at least partially Ca++ sensitive, whereas the binding to the 40,000 Mr protein does not. 125I-G-actin bound to proteins in the SDS gels can be fixed in situ and compared directly with the stained gel. This technique should prove generally useful in identification and purification of some actin-binding proteins from cells and tissues.
D000199:Actins / Q000097:blood* / Q000378:metabolism; D001792:Blood Platelets / Q000032:analysis*; D002118:Calcium / Q000494:pharmacology; D002352:Carrier Proteins / Q000097:blood*; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005404:Fixatives; D018260:Gelsolin; D006801:Humans; D007457:Iodine Radioisotopes; D008840:Microfilament Proteins; D012394:Rosaniline Dyes
D000199:Actins; D002352:Carrier Proteins; D005404:Fixatives; D018260:Gelsolin; D007457:Iodine Radioisotopes; D008840:Microfilament Proteins; D012394:Rosaniline Dyes; C016953:brevin; D004533:Egtazic Acid; C004692:coomassie Brilliant Blue; D002118:Calcium